Immunostaining
F Ulrich Hartl, Cole S Sitron, Victoria A Trinkaus
Abstract
This is a protocol that describes how to use antibody staining to detect cellular epitopes by immunofluorescence microscopy.
Attachments
Steps
Fixation
Aspirate media and gently wash each well with 1X PBS.
Aspirate PBS and place 500µL of 4% paraformaldehyde onto each well.
Fix at Room temperature for 0h 10m 0s.
Remove 4% paraformaldehyde and add 1mL 1X PBS.
Immunostaining
Permeabilize 1mL 0.1% Triton X-100 and gently shake at Room temperature for 0h 5m 0s.
Block in 1mL Blocking Buffer for 1h 0m 0s.
Dilute primary antibodies into Blocking Buffer and add 300µL of diluted antibodies to each well.
Shake at 4°C 1h 0m 0s.
Wash 3x with 1mL PBS, gently shaking at Room temperature each time for 0h 5m 0s.
During washes, centrifuge tubes of secondary antibodies at 16000x g,0h 0m 0s for 0h 10m 0s at 4°C.
Dilute secondary antibodies into Blocking Buffer 1:500.
Incubate coverslips in 300µL diluted secondary antibody for 2-3 hr at Room temperature with gentle shaking, protected from light.
Wash with 1mL PBS for 0h 5m 0s with gentle shaking.
Add 1mL diluted NucBlue and incubate with gentle shaking for 0h 5m 0s, protected from light.
Wash 2x with 1mL PBS for 0h 5m 0s with gentle shaking.
Allow fluorescence mounting medium to come to Room temperature.
Add one drop of fluorescence mounting medium onto microscope slides for each coverslip.
Mount coverslips onto slides.
Let slides cure at Room temperature 0h 5m 0s, protected from light.
